Experiment 8: Enzymatic Methods of Carbohydrate Analysis Introduction Carbohydrates are the most abundant members of the large and

important class of biomolecules. Biologists believe that the carbohydrates make up a higher percentage of biomass than any other biomolecule class. Carbohydrates are naturally occurring compounds that have reactive aldehyde or ketone functional groups as well as multiple hydroxyl groups. It can be found in all forms of life and serve many different functions. The carbohydrates are best known for their roles in energy metabolism, structural roles and cell communication. The most abundant carbohydrates in nature are the polysaccharides starch, glycogen, and cellulose. Carbohydrates in the simplest form are known as monosaccharides or simple sugars such as glucose, fructose, and galactose, which hav the empirical formula e (CH2 O)n. Glucose and fructose (C6H12O6) function as energy sources in cells during cellular respiration. Disaccharides are formed by joining of two monosaccharides through hydrolysis. Maltose is one of the most abundant disaccharides in nature. It is a product from the hydrolytic breakdown of starch. Polysaccharides are composed of many monosaccharide units. Plants store excess glucose in the form of starch. Animals (and humans) store excess glucose in the form of glycogen in the liver and muscles. Other important polysaccharides are cellulose and chitin. Cellulose makes up the cell wall of plants whereas chitin provides structure to fungi and the exoskeleton of arthropods. Enzymatic method can yield maximum specificity for glucose estimation. Glucose can be measured by its reaction with glucose oxidase, in which gluconic acid and hydrogen peroxide are formed. Hydrogen peroxide than reacts with an oxygen acceptor, such as ortho-dianisidine, phenylamine-phenazone or any other

chromogenic oxygen acceptors, in a reaction catalysed by peroxidase to form a colour. One of the advantages of enzymatic method is its specificity and their ability to catalyze specific reactions. This enables the estimation of glucose even in a complex mixture. Thus, this method is widely applied in the field of clinical chemistry and for food analysis. In clinical chemistry, the enzymatic analysis of glucose in blood and urine samples has been modified as dipstick test. Glucose oxidase is the most widely employed enzyme as analytical reagent. This is the result of its utility in the

determination of glucose, an analyte of wide analytical interest, and its relatively low cost and good stability that make the glucose/glucose oxidase system a very convenient model for method development (particularly in the area of biosensors). Objectives 1. To analyse the carbohydrate by using enzymatic method. 2. To understand the principles of enzymatic estimation of glucose.

Apparatus and Materials 1. Starch 3. -Amylase 2. Anhydrous glucose 4. Amyloglucosidase (AMG) 6. Glucose oxidase 8. Whatman filter paper 10. Water bath 12. Cuvettes 14. Micropipette

5. AMG Buffer 7. Ice 9. Aluminium foil 11. Test tubes 13. Spectrophotometer

Methods A) Digestion with AMG 1) 0.2mL of -amylase added to 100mL of purified water to prepare blank. 2) Fresh 1 mg/mL glucose standard were make up from anhydrous glucose to prepare glucose standard. 3) 1mL of the above prepared sample were taken to prepare samples. 4) The solutions according to the table below were pipetted into test tubes and label properly. Adjust volume Blank -amylase Glucose (1mg/mL) Samples 1mL 1mL 1mL AMG 1mL 1mL AMG buffer 1mL -

5) All the pipetted solutions were gently mixed, and then is incubated in a 60 C water bath for 25 minutes. C.

6) After that, all the pipetted solutions were cooled to 20

7) Exactly 8mL of water were added to each of the tubes. 8) The samples were filtered through Whatman filter paper. 9) Total of 1mL from the AMG digestion were taken for analysis of total glucose. B) Analysis of total glucose 1) According to the table below, these tubes were pipetted into screw capped tubes. These tubes were labelled accordingly. 2) These tubes were placed in a water bath 35 C for 45 minutes. Each tubes

were covered with aluminium foil to keep protected from the light. 3) Then the samples were cooled to room temperature for 10 minutes in the dark. 4) The absorbance in 1-cm cells at 505nm were measured against the water blank . 5) The absorbance is read within 30 minutes. 6) The -amylase reading is subtracted from the sample reading . 7) The amount of glucose in the samples was determined by comparing them to the standard curve. 8) The amount of glucose in the samples was multiply by 0.9 to give an indication of the amount of starch in the samples. This is an adjustment from free glucose to anhydrous glucose as occurs in starch.

Results Analyte Blank water Blank amylase Samples Glucose 10g Glucose 20g 20L 980L 5mL 0.025 20.0g 1mL 10L 990L 5mL 5mL 0.702 0.009 652.8g 10.0g 1mL Volume Water 1mL GOP 5mL 5mL Abs 505nm 0.000 0.071 Concentration

Glucose 40g Glucose 80g Glucose 100g Glucose 160g

40L

960L

5mL

0.016

40.0g

80L

920L

5mL

0.063

80.0g

100L

900L

5mL

0.036

100.0g

160L

840L

5mL

0.053

160.0g

Discussions A graph of absorbance versus concentration of glucose is plotted based on the results obtained. Based on the graph obtained, the absorbance is directly proportional to the concentration of glucose. As the concentration of the glucose increases, the glucose content increases, there are more molecules in that solution that causes the turbidity of the glucose solution to increase. When there are more molecules in the solution due to the increased concentration, the amount of transmitted light decreases. Thus, this increases the absorbance of the glucose solution. Based on the absorbance obtained, a calibration curve is prepared from standard solutions (solutions of known concentration) and the concentration of the sample which is 726.2g is obtained by using the formula of
.

However, there are some inaccurate of results obtained for glucose with concentration of 40g and 80g. This may due to some errors we had made during the experiment. Hence, there are some precautions that must be taken during this experiment. First of all, parallax error should be avoided when measuring the solution by taking the reading at the level perpendicular to the surface. Next, aluminium foil must be used to protect the analytes together with GOP as the glucose oxidaseperoxidase reagent (GOP) is light sensitive. Any deviation of results may be obtained if GOP is exposed to the light. The observing of the solutions temperature time to time is also very important as their temperatures may fluctuate very fast. So measuring of the absorbance of the solutions must be carried out as soon as possible once they are taken out from the hot water bath or ice cubes beaker to obtain more

accurate results. In this experiment, the temperature is the main factor causing inaccurate results obtained. Alpha-amylase is an enzyme that hydrolyses alpha-bonds of large alpha-linked polysaccharides such as starch and glycogen, yielding maltose and glucose. It is the major form of amylase found in humans and other mammals for the process of digestion. In this experiment, alpha-amylase reading is required to be subtracted from the sample reading of absorbance value. This is because glucose is locked up in many larger forms, including lactose and sucrose where two small sugars are connected together and long chains of glucose like starches and glycogen and alpha-amylase is used to break these chains into their individual glucose units. Hence, this means that the sample also contains small amount of alpha-amylase used for the breaking down of complex molecules into glucose units and a more accurate result of absorbance value of the sample for determining the concentration of glucose could be obtained by subtracting the absorbance value of alpha-amylase that may contain in the sample that would lead to a deviated result. Alpha-amylase contained in the sample may affect the turbidity of the solution and hence affected the absorbance value obtained. Whatman filter papers are used in this experiment to filter the samples before withdrawing of 1mL for the second part of the experiment. They have a high wet strength due to the addition of a small amount of chemically stable resin. Normal qualitative applications will not introduce any significant impurities into the filtrate but not Whatman filter paper. Filter paper is used to rid a liquid of solid particles. It comes in different sizes and grades. Water bath is a device used for regulating the temperature of substances subjected to heat. The vessel is surrounded by another vessel containing water which can be kept at a desired temperature. Water bath is frequently used in chemistry labs for a number of temperature related applications. Water baths are used to heat those substances, which cannot be heated directly on Bunsen burner or hot plate or any other media. However, only material with boiling point less than that of water bath could be heated using water bath.

Conclusion In conclusion, the absorbance value is directly proportional to the concentration of glucose. As the glucose content increases, the absorbance increases. References 1. Universiti Tunku Abdul Rahman. Structural Biochemistry Lab Manual Version 01-08. Bachelor of Science (Hons) Biomedical Science. Page 68-71. Retrieved August 17, 2010. 2. Rodney Boyer, Concepts in Biochemistry, 3rd edition, 2006, John Wiley & Sons, Inc. Retrieved August 17, 2010. 3. Filter Paper. Retrieved August 17, 2010, from

http://www.newton.dep.anl.gov/york/filter.html.